This invention relates to a composition and a method for isolating or immobilizing polynucleotides and polypeptides by means of aromatic derivatives of certain carbohydrates and, in particular, triphenylmethyl ether derivatives of polysaccharides.
The employment of a variety of support materials to attract and strongly bond substances such as nucleic acids and proteins has advanced research endeavors in the field of biological chemistry. The physical/chemical forces between such substances and supports is generally attributable, either exclusively or in combination, to such mechanisms as covalent attachment, ionic bonding, hydrogen bonding, hydrophobic bonding, adsorption, and physical entrapment.
Special support materials are used to isolate and purify polynucleotides such as RNA or DNA for research purposes. In this regard, for example, isolation of poly A-containing mRNA and denatured DNA are currently very important manipulative procedures in biochemistry/molecular biology. Furthermore in the field of biocatalysts, proteins in the form of specific enzymes are joined to, and sometimes stabilized by, support materials. Such activated materials are now gaining favorable commercial acceptance. As is generally known, there are several advantages accruing from the binding of enzymes to suitable supports:
(1) Given a reasonable stability of the immobilized enzyme, one can reuse the enzyme a number of times with considerable saving of time and money;
(2) Ease and speed of separating the products of a given reaction from the immobilized enzymatic catalysts; and
(3) Replacing routine chemical synthetic methods with enzyme-mediated reactions, as in the synthesis of penicillin derivatives, with attendant improvements in the yield, speed, and stereochemical purity.
The particular type of chemical linkage, covalent or non-covalent, joining the enzyme to the support may conveniently be used to categorize and distinguish between the various types of enzyme immobilization/stablization methods. Generally speaking, enzymes may be immobilized to solid matrices by means of non-covalent or covalent chemical bonds. In the case of non-covalent bonds, conditions being very mild and uncomplicated, the ultimate enzyme attachment yields may be quite high; furthermore, little time or skill is required. One general drawback of this approach is that, for a given enzyme, conditions optimal for catalytic efficiency may not coincide with conditions optimal for irreversible immobilization of the enzyme to the support, and as a result, leakage may ensue. In the case of covalent bonds linking an enzyme with its solid support, the chemical requirements to effect this linkage may be excessively harsh, as far as pH, salt, or organic solvent requirements are concerned, so as to seriously denature or inactivate many fragile enzymes. Covalent bonds once established between an enzyme and support are stable, although recent investigators find unexpected instability for some apparently widely used covalent attachment methods. Given the nature of the chemistry employed, considerable skill is required to do these reactions well. For the reason just mentioned, covalent attachment methods, while adequate with some enzymes, have their limitations and often fail to provide a very broad-based generalized method of enzyme immobilization.
With regard to covalent methods of attaching enzymes to support materials, one general approach advocated by workers in the field involves the formation of amide or related bonds between certain amino groups of the enzyme and some activated form of the support involving azide, isocyanate, carbodiimides, or iminocarbonates. A further approach has been methods involving the use of diazo coupling agents. In this regard, U.S. Pat. No. 3,647,630 to Franks discloses a preparation of a diazotized anthranilate ester of cellulose. Another procedure uses Schiff's base techniques. U.S. Pat. No. 3,947,352 to Cuatrecasas et al. discloses affinity chromatographic techniques using intermediate Schiff bases formed from sodium metaperiodate with polysaccharides. Another class of reagents found useful are the halides and sulfates of titanium, tin, zirconium, or iron derivatives of polysaccharides as set forth in U.S. Pat. No. 3,841,969 to Emery et al. In this general category also are the various cyanogen halide activated polysaccharides as disclosed in U.S. Pat. Nos. 3,914,183 to Johansson et al. and 4,167,446 to Huper. Furthermore, substantial research has been conducted with respect to ester types of linkages with polysaccharides and their derivatives to achieve activation thereof. In this regard, U.S. Pat. No. 3,909,360 to Horiuchi et al. discloses water-insoluble carrier-bound enzymes, formed by the use of fatty acid esters of polysaccharides. U.S. Pat. No. 3,833,555 to Keys et al. discloses a polysaccharide cyclic carbonate containing compounds, the compositions being useful for insolubilizing enzymes. A further process for binding biologically active proteins in an aqueous phase is disclosed in U.S. Pat. No. 4,038,140 to Jaworek et al., wherein the use of activated polysaccharides is described having a hydrophilic graft co-polymer grafted thereinto. Further, U.S. Pat. No. 3,278,392 to Patchornik discloses the use of bromacetyl cellulose having bonded thereto enzymes through sulphhydryl groups or amino groups of the enzymes.
With regard to non-covalent chemical attachment methods, simple adsorption of a given enzyme to some solid support is occasionally employed. In practice, this approach may be very limited in that any changing of the reaction conditions for the adsorbed enzyme can have a rather unpredictable and often weakening effect on the relative tightness of the adsorption. Adsorption of some enzymes onto ion-exhange columns such as diethyl aminoethyl cellulose is occasionally used in laboratory work. The efficacy of this approach depends on a somewhat fortuitous situation in that at a given pH where an enzyme is maximally active, the celulose derivative must possess a rather high net positive charge. Water-insoluble aromatic tannin preparations are disclosed by Chibata et al. in U.S. Pat. No. 4,090,919; in this instance, enzymes are apparently immobilized through a combination of ionic and hydrophobic bonds, U.S. Pat. No. 4,006,059 to Butler discloses a hydrophobic non-covalent binding of proteins to support materials. Various hydrophilic solid supports such as cellulose or glass have been esterified with phenoxyacetyl groups beginning with the corresponding acid chlorides. Finally, benzoylated cellulose substrates have been used for the isolation of poly adenylic acid containing RNA and other polynucleotides, its use being described in the open literature, Biochemistry, Vol. 13, No. 18, pp. 3677-3682, 1974.